Transcriptomic And Proteomic Profiles Of SSN-1 Cells Infected With SHVV With An Emphasis On Virus Replication Related Factors

Rhabdovirus is a kind of single-stranded,negative-sense enveloped RNA virus with rod-or bullet-shape.The nucleocapsid of the virus consists of genomic RNA,nucleoprotein(N),RNA dependent RNA polymerase(L)and phosphoprotein(P).In addition,the nucleocapsid is coated with matrix protein and glycoprotein which forms the spike on the viral surface.Rhabdoviridae consists of more than 185 different viruses isolated from various hosts,including plants,insects,fish and mammals,and cause serious diseases of the infected hosts.More than twenty species of fish rhabdoviruses have been reported.They can cause great economic loss in aquaculture.Currently,there is no effective prevention and control strategy for fish rhabdovirus infections.Understanding the host factors in response to virus infection is the basis for the prevention of rhabdovirus infection.In the present report,a new rhabdovirus was isolated from diseased hybrid snakehead fish.Viral genomic RNA nucleotide sequence analysis of the isolated virus strain showed that it belonged to the genus of Perhabdovirus and it was named snakehead fish vesiculovirus(SHVV).In this study,the transcriptomics and proteomics technologies were applied to reveal the the interactions between the virus and the host.We also carried out the research on host ranges and viral proteins expressed in baculovirus expression systems as well as prokaryotic expression system.The obtained results were as follows:1.We showed that SHVV was able to replicate and proliferate very well in SSN-1 cells which originated from striped snakehead fish(Channa striatus)and it caused obvious cytopathic effects in the cells SHVV showed high genomic RNA sequence homology with Siniperca chuatsi rhabdovirus(SCRV)and Monopterus albus rhabdovirus(MoARV).SHVV encodes five viral structural proteins:Nucleoprotein(N),Phosphoprotein(P),Matrix protein(M),Glycoprotein(G)and RNA dependent RNA polymerase(L).There have been shown that G protein of rhabdoviruses is a good antigen for vaccine development.Therefore,baculovirus expression system was used to generate recombinant baculoviruses which could efficiently express SHVV-G protein.In doing so,we transfected the DNA Sapphire baculovirus DNA and the ProGreen Baculovirus DNA into the sf9 insect cells to generate the recombinant baculovirus to express SHVV His-tag and GFP-tag fused G proteins.We also succeeded to express the SHVV N protein and P protein using prokaryotic expression system in Escherichia coli.Polyclonal antibodies against N and P proteins were genertated by injection of the recombinant proteins into rabbit and mouse,respectively.Taking the advantage of the generated antibodies,we showed that the N and P proteins were mainly distributed in the cytoplasm of SSN-1 infected with SHVV.Moreover,SHVV could infect mandarin fish and snakehead fish.The infected fish showed typical clinical signs of rhabdovirus infection,including haemorrhage and oedema.Histopathological analysis revealed that extensive inflammation and necrosis were observed in the spleen,kidney and liver of the moribund mandarin fish.2.In this study,the transcriptomic profile of SHVV-infected or mock-infected SSN-1 cells at 3 and 24 hours(h)post of infection(poi)were obtained using high-throughput sequencing technique.A total of 93,372 unigenes were obtained and 18870 unigenes of them were annotated in NR database(20.1%).The differently expressed genes(DEGs)of SSN-1 cells upon SHVV infection were thereby identified,including 3,668 and 3,536 DEGs at 3 and 24 h poi,respectively.In 3 h group,there were 2867 up-regulated GEGs,801 down-regulated DEGs.In 24 h group,there were 2468 up-regulated DEGs and 1068 down-regulated DEGs.The DEGs were further annotated using GO,KOG and KEGG databases.The results showed that these DEGs were involved in many pathways of viral pathogenesis,including retinoic acid-inducible gene I(RIG-I)like receptors pathway,Toll-like receptor signaling pathway,NF-kappa B signaling pathway,PI3K-Akt signaling pathway,and MAPK signaling pathway etc..Therefore,several immune-related DEGs were randomly selected and confirmed by quantitative real-time PCR(qRT-PCR).The qRT-PCR results showed similar expression patterns with those of transcriptomic results,indicating that the transcriptomic profiles were reliable.3.As mentioned above,a large number of DEGs were identified.We further focused our analysis on the host anti-viral immunity.From the transcriptome results,the IFN-I pathway was triggered by SHVV infection,resulting in the up-regulation of many interferon-stimulated genes(ISGs).Among them,interferon inducible protein 35(IFI35)was significantly up-regulated during SHVV infection.To investigate the role of IFI35 in SHVV infection,we amplified the open reading frame of IFI35 and generated a plasmid pEYFP-IFI35.In SSN-1 cells,IFI35 was either knock-downed using its specifc siRNA or IFI35 was over-expressed by transfection of pEYFP-IFI35.Subsequently,the treated SSN-1 cells were infected with SHVV.The mRNA levels of viral genes were increased when IFI35 was over-expressed,while they were decreased when IFI35 was knock-downed,indicating that IFI35 could facilitate the viral mRNA transcrition.To further address the mechanism underlying function of IFI35,the mRNAs of genes related to RIG-I like receptors pathway,such as RIG-I,MAVS,TRAF3,IRF3 and IRF7 were quantified by qRT-PCR,the results showed that they were all up-regulated,indicating that SHVV replication maybe regulated by the RIG-I like receptors pathway associated with IFI35.4.Proteomic analysis of SHVV infected SSN-1 cells were performed to better understand the host and SHVV interactions.A total of 15619 peptides matching 2249 proteins were obtained using iTRAQ technique.Differently expressed proteins(DEP)were further analyzed,the results showed that there were 85 DEPs(48 up-regulated and 37 down-regulated)and 422 DEPs(232 up-regulated and 190 down-regulated)were identified at 3 h and 24 h poi,respectively.Gene ontology analysis and the KEGG database were employed to analyze the pathways of the DEPs.Since transcriptomic profiles of SHVV infected SSN-1 cells were employed above,the correlations between transcriptomic and proteomic results were further explored.The results showed that there were 6 common DEGs and DEPs(5 up-regulated and 1 down-regulated)and 31 common DEGs and DEPs(24 up-regulated and 7 down-regulated)were identified at 3 h and 24 h poi,respectively.Part of the common DEGs were confirmed by qRT-PCR.Moreover,the protein-protein interaction analysis network showed that SHVV replication was closely related with the Toll-like receptor signaling pathway,RIG-I-like receptor signaling pathway,MAPK signaling pathway and Herpes simplex infection pathway.5.MicroRNAs(miRNAs)play important roles in mediating multiple biological processes in eukaryotes and are being increasingly studied to evaluate their roles associated with cellular changes following viral infection.To identify specific miRNAs involved in SHVV infection,we performed microRNA deep sequencing on SSN-1 with or without SHVV infection.A total of 205 known miRNAs were identified when they were aligned with the known zebrafish miRNAs,and 9 novel miRNAs were identified using MiRDeep2 software.18 and 143 of the 205 known miRNAs were differentially expressed at 3 and 24 hours poi,respectively.From the differentially expressed miRNAs(DE-miRNA),5 were randomly selected to validate their expression profiles using qRT-PCR,and their expression profiles were consistent with the results of the microRNA sequencing.In addition,the DE-miRNA target genes were predicted and they covered a wide range of functions.Some of the target genes were closely related to immune regulations,such as mitochondrial antiviral-signaling protein,Toll-like receptor 9 and interferon regulatory factor 3.The resulst showed that there were 10 and 58 DE-miRNAs were predicted to bind to SHVV genomic RNA at 3 and 24 h poi,respectively.The effects of three selected miRNAs(miR-130-5p,miR-214 and miR-216b)on SHVV multiplication were evaluated using their mimics and inhibitors via qRT-PCR and Western blotting.The results showed that miR-214 and miR-130c-5p were able to inhibit the replication of SHVV,while miR-216b didn’t show any effect on virus replicationOur findings shed a new light on the interactions between SHVV and host,and will pave a new way for the developing novel strategies against SHVV infection.